Development of novel DNA vaccine and immunotherapeutic approaches

Abstract: Many potential DNA vaccination and immunotherapeutic strategies has been explored in both animal models and human settings in recent years. However, the failure of efficient and safe method development suggests that it is time to rethink, since more innovative approaches are needed for better control of infection and immunotherapy. DNA-based vaccination is a relatively new technology. Such a vaccine consists of a circular piece of DNA (plasmid), which contains an immunity-inducing gene. Once inside a cell, the DNA vaccine vector produces the protein antigen, which is seen as foreign and leads to the generation of an immune response. However, DNA vaccination has so far met with limited success likely due, at least in part, to the lack of strong elicited immune responses in humans. There is therefore a need for the development of appropriate adjuvant, which can be used together with DNA vaccines. Bioplex technology is a novel non-viral approach, which makes it possible to overcome some of the barriers for efficient delivery of antigen encoding plasmid DNA into the cell. In order to incorporate different functional entities into plasmid DNA for specific purposes, highly stable anchors of either Locked nucleic acid (LNA) or bisPNA (Peptide nucleic acid) can be used to attach several and different functional entities. In the first paper, we investigated the possibility of stabilizing sequence-specific binding of PNA-anchored functional peptides to plasmid DNA by hybridizing PNA and LNA oligomers as "openers" to partially overlapping sites on the opposite DNA strand. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification. In the second study, we developed a novel branching technique to generate multiple anchor binding sites per plasmid. The LNA oligonucleotides were synthesized with an extension of bases that is not used for plasmid hybridization, but designated for hybridization to secondary oligonucleotides. This universal anchor system is versatile and can give an exponential growth of number of functional entities presented per plasmid. This new branching approach is very effective for attaching nucleic acid-based toll-like receptor (TLR) ligands for DNA vaccination and immunotherapy. In third study, we wanted to determine the potential role of Bruton's tyrosine kinase (Btk) in the TLR signaling pathway in mouse splenic B cells. Inhibition of attenuators represents a generally applicable and alternative strategy for enhancing the potency of various forms of prophylactic and therapeutic vaccines. Our data provide evidence in support of the theory that Btk negatively regulates both TLR9 activation and expression in mouse splenic B cells. Thus, down regulation of Btk with plasmid expressing short hairpin RNA (shRNA) and attaching TLR9 ligands in the same construct might be a powerful tool for enhancing immune responses. In the fourth study, we investigated the effect of simultaneous administration of TLR3 ligand (poly I:C) and single stranded DNA oligonucleotides (ssDNA ODN) on human monocytederived dendritic cells (moDCs). Using poly I:C as a DC maturing agent, we show that the maturation and cytokine production can be significantly inhibited by ssDNA ODN. The addition of ssDNA together with poly I:C blocks phosphorylation of IRF3, a downstream component of this pathway. Taken together our findings indicate that ssDNA inhibits some yet to be identified pathway(s) leading to maturation of DCs that might be promising candidates for the development of novel immunotherapeutic approaches. In conclusion, the present work describes the development of new technologies for vaccination and immunotherapy. The ideal non-viral technological platform might include an immune cell-targeting molecule, shRNA construct against negative regulators of immune signaling and addition of TLR agonists in the same setting. Through this work we have developed a strategy, which could serve as an embryo for future development.